We have examined the intra-thymic differentiation of functionally and phenotypically distinct T cell subsets as well as their interaction with thymic epithelium. Studies on thymocytes from genetically defective scid mice have suggested that TcR+ cells play a critical role in promoting the entry of thymocytes into the CD4/CD8 differentiation pathway as well as in promoting the maturation and organization of thymic medullary epithelium. Phenotypic studies on developing thymocytes have identified two distinct, but inter-related subsets of thymocytes that express identically skewed TCR repertoires, namely CD4-CD8-TCR- alpha-beta+ thymocytes and Ly6C+ thymocytes. Ly6C+ thymocytes were found to represent a readily identifiably subpopulation within each CD4/CD8 thymocyte subset; nevertheless, the Ly6C+ thymocytes within each CD4/CD8 thymocyte subset expressed a distinctive TCR repertoire marked by overexpression of V-beta8 and expression of autoreactive TCR. Finally, we found that thymocytes readily acquire surface CD4 and CD8 determinants from other thymocytes, demonstrating that caution is necessary in using low level CD4/CD8 expression to identify novel thymocyte subsets.